JPH0633259B2 - Method for separating and purifying berberine alkaloids - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for separating and purifying berberine alkaloids from a berberine alkaloid-containing material. Berberine alkaloids are compounds in demand as intestinal medicines, therapeutic agents for bacterial enteropathy, yellow dyes and the like.

[Prior Art] Regarding a conventional method for separating a berberine alkaloid from a berberine alkaloid-containing material, a plant that produces a berberine alkaloid, for example, a tissue of a plant such as yellow oak belonging to the Rutaceae family, orensis belonging to the buttercup family, or the tissue Several methods have been reported for separating and purifying berberine from callus obtained by culturing as a raw material.

For example, in Japanese Patent Application Laid-Open No. 47-30897, callus obtained by tissue culture is crushed and extracted with methanol, and the extract is concentrated and then hydrochloric acid is added to acidify it, followed by ether extraction. A method is disclosed in which the ether-insoluble portion is made alkaline with ammonia, extracted with chloroform, and the chloroform-soluble portion is concentrated and subjected to thin layer chromatography (TLC) to fractionate berberine.

Further, JP-A-50-13519 and JP-A-51-12993 disclose that yellow callus callus obtained by tissue culture is crushed and extracted with hot water or methanol. After concentration under reduced pressure, acidification with hydrochloric acid was performed to perform column chromatography using methanol as an eluent and alumina to remove impurities, and the resulting berberine-containing methanol solution was homogenized by adding hot water and then chlorinated by cooling. A method for depositing and separating berberine is disclosed.

Also, in Japanese Patent Laid-Open No. 55-155056, 95% of ground sardines are used.
Extraction is performed by adding ethanol, and then the extract is concentrated, water and talc are added to the extract, and the mixture is extracted by heating and then filtered to remove impurities, and the berberine-containing pigment extract is extracted. Is disclosed. The method is a method for obtaining a berberine extract, and the publication does not disclose any method for isolating berberine.

Further, Japanese Patent Application Laid-Open No. 57-144992 discloses a method for separating gerberoline alkaloids of diatrrolidine and palmatine from a culture obtained by tissue culture of a plant of the family Astragalus. According to this method, the culture is separated into culture cells and culture medium by filtration, and then air-dried culture cells are extracted with ethanol, while the culture medium is made alkaline and then extracted with chloroform. After concentration, this was developed by thin layer chromatography (TLC) to fractionate the spot containing the target component, and this was made into an aqueous solution, then potassium iodide was added to precipitate it as an iodo salt, and the desired berberine alkaloid was prepared. A method of separating the is shown.

All of the conventional methods for separating and purifying berberine alkaloids described above are very complicated in many operation steps, and the recovery efficiency of the berberine alkaloids to be obtained is low,
In addition to being inefficient, various fatty acid esters, which are impurities during the extraction, are associated with it, so that it is necessary to perform a degreasing treatment with an ether treatment afterwards.

As a new separation method different from the above method, the applicant has
According to Japanese Patent Application No. 59-82951, a culture mixture comprising cultured cells and a culture solution obtained by tissue culture of Japanese red pine,
Alternatively, chlorine ions or nitrate ions are added to the culture solution containing berberine alkaloids released from the culture cells into the culture solution to precipitate the berberine alkaloids as slightly soluble yellow crystals, which are isolated and recrystallized as necessary. Then, a method for separating and purifying berberine alkaloids was shown.

[Problems to be solved by the invention]

The present inventors have examined conventional methods for separation and purification of berberine alkaloids, and as for all other methods except Japanese Patent Application No. 59-82951, as described above, there are various operation steps, such as complicated and low recovery efficiency. It is admitted that it has the defect of. On the other hand, in the method of Japanese Patent Application No. 59-82951, the berberine alkaloid-containing aqueous solution released from the cultured cells into the liquid medium during the culture and dissolved therein, or the cultured cells was crushed to extract the berberine alkaloids outside the cells. This is a method in which a berberine alkaloid-containing aqueous solution is used as a raw material and chlorine ions or nitrate ions are added thereto to precipitate berberine alkaloids, but further improvement is desired. For example, a method is desired in which cultured cells do not necessarily have to be mechanically disrupted, and the purity of the separated berberine alkaloid can be increased.

Under such circumstances, the present inventors have, unlike the conventional method, separated a berberine alkaloid from a berberine alkaloid-containing material with a high purity by a simple method with a small number of steps and with a high recovery rate. The method for purification was earnestly studied.

[Means and Actions for Solving Problems]

As a result, they have found that the above object can be achieved by adopting the following method, and completed the present invention.

That is, according to the method of the present invention, the formula (I) Berberine represented by (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄
= -OCH ₃ ), palmatin (R ₁ , R ₂ , R ₃ , R ₄ = -OC
H ₃ ), Copteisine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄ =-
OCH ₂ O-), Jatrolidine (R ₁ = -OH, R ₂ , R ₃ , R ₄
= -OCH ₃ ), Columbamine (R ₁ , R ₃ , R ₄ = -OCH ₃ , R ₂
= -OH), salif engine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ =-
OCH ₃ , R ₄ = -OH) is added to the extract obtained by treating plant tissue pieces or cells containing an isoquinoline alkaloid selected from OCH ₃ and R ₄ = -OH with sulfuric acid, and a salt exchange agent containing chloride ion and / or nitrate ion. A method for separating and purifying an isoquinoline alkaloid, which comprises depositing an isoquinoline alkaloid as a salt of the ion and separating the salt.

Specific examples of tissue pieces or cells of a plant containing berberine alkaloids used as a raw material in the method for separating and purifying berberine alkaloids of the present invention are Oren, buttercup family plants such as pine, Rutaceae plants such as yellowfin, etc. Is a tissue piece of a berberine alkaloid-producing plant or a cultured cell (including callus) obtained by tissue culture of the plant tissue piece. Since the cultured cells are usually a mixture of the cultured cells and the medium, for example, when a liquid medium is used as the medium, the culture mixture is divided into the cultured cells and the culture solution as needed. The cultured cells containing the berberine alkaloid can be used as a raw material for the subsequent extraction operation of treating with the sulfuric acid of the present invention described later in a wet state or after being dried by air drying or the like. On the other hand, in the culture medium, berberine alkaloids may be partially released from the cultured cells to the outside of the cell, but in this case, the cultured cells and the liquid medium are separated and the aqueous solution obtained by treating the cultured cells with sulfuric acid described below is liquid. The medium may be combined with a salt exchange agent to be described later, or the whole culture mixture may be treated with sulfuric acid to extract the berberine alkaloids inside the cells into an extracellular aqueous solution, Next, a method of adding a salt exchange agent described below to the berberine alkaloid-containing aqueous solution after removing the cell debris in the culture mixture treated with sulfuric acid by filtration or the like may be adopted.

Specific examples of the berberine alkaloids separated and purified by the method of the present invention include compounds of the formula [I] Berberine represented by (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄
= -OCH ₃ ), palmatin (R ₁ , R ₂ , R ₃ , R ₄ = -OC
H ₃ ), Copteisine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄ =-
OCH ₂ O-), Jatrolidine (R ₁ = -OH, R ₂ , R ₃ , R ₄
= -OCH ₃ ), Columbamine (R ₁ , R ₃ , R ₄ = -OCH ₃ , R ₂
= -OH), salif engine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ =-
OCH ₃ , R ₄ = -OH) and other isoquinoline-based alkaloids. Of these, berberine is preferred in the present invention.

In the present invention, the above-described berberine alkaloid-containing material is treated with sulfuric acid to obtain an extract containing berberine alkaloid. A sulfuric acid aqueous solution is usually used as the sulfuric acid as the extractant in this case, and the sulfuric acid concentration at this time is usually in the range of 0.1 to 3 N (N), preferably 0.2 to 0.5 N. In the present invention, the amount of the extractant used is usually 1 to 5 times by weight, preferably 3 to 4 times by weight, of the sulfuric acid aqueous solution in the above concentration range per 1 part by weight of the berberine-containing substance. The extraction is carried out at a temperature of 10 to 50 ° C. for about 1 hour or more and usually with stirring. After the extraction treatment, the extraction residue containing the berberine alkaloid is obtained by removing the extraction residue of the solid matter by filtration or the like by a usual method.

In the present invention, the extract containing the berberine alkaloid obtained by the above-mentioned method has a chloride ion or /
And by adding a salt exchange agent containing nitrate ions, the berberine alkaloids are precipitated as salts of the ions.

In the present invention, when a salt exchange agent is added to the above-mentioned extract, this extract may be concentrated to a solution containing an appropriate concentration and containing no solid matter, if necessary. The salt exchange agent used in the present invention is a water-soluble compound containing chloride ion and / or nitrate ion, and specific examples thereof include hydrogen chloride, hydrochloric acid, lithium chloride, sodium chloride, potassium chloride, nitric acid and lithium nitrate. , Sodium nitrate, potassium nitrate and the like. In the present invention, it is preferable to use hydrogen chloride, hydrochloric acid or nitric acid among them, and in this case, hydrochloric acid or nitric acid may be an aqueous solution having an appropriate concentration, but usually has a concentration of 3 to 14.5 N. Is used.

In the present invention, when the salt exchange agent is added to the extract, the amount of chloride ion and / or nitrate ion is usually 10 to 40 times by mole with respect to 1 mole of berberine alkaloid in the extract. , Preferably 20 to 30 times by mole. In the present invention, by selecting the addition amount of the salt exchange agent in this way, the berberine alkaloid in the extract can be efficiently converted to the hydrochloride or nitrate of the berberine alkaloid, which is hardly soluble in the extract, to cause precipitation. You can In this case, the temperature for precipitation is usually 0.
To 50 ° C, preferably 10 to 30 ° C. In the present invention, the berberine alkaloid in the solution is thus converted into a sparingly soluble salt for precipitation, and the precipitate is separated from the solution by a usual method such as filtration.

In the present invention, the berberine alkaloid hydrochloride or / nitrate separated from the solution by the above-mentioned method is, for example, water,
It is washed with a cleaning agent such as water-containing methanol, water-containing ethanol, methanol, ethanol or acetone. After the washing, a berberine alkaloid can be obtained as a highly pure hydrochloride or nitrate of the alkaloid by performing a purification operation such as recrystallization using a recrystallization solvent such as a lower aliphatic alcohol such as methanol or ethanol an appropriate number of times. You can

〔The invention's effect〕

According to the method of the present invention, a berberine alkaloid can be separated and purified from a berberine alkaloid-containing material with a high purity and a high recovery rate by a simple method having fewer steps than the conventional method.

〔Example〕

Hereinafter, the method of the present invention will be specifically described with reference to Examples.

Example 1 In an extraction tank equipped with a stirrer, 6.7 kg of raw calli of oren obtained by the tissue culture method (berberine content: 2.0%),
14.7 kg of 0.3N sulfuric acid was added, and the mixture was stirred at 25 ° C. for 3 hours and then filtered. 550 g of NaCl was added to this filtrate while stirring at room temperature,
Stirring was continued for 3 hours. The resulting crude berberine chloride precipitate was filtered off, washed with water and dried. 110 g of this crude berberine chloride was dissolved by heating in 900 g of methanol, and the insoluble matter was filtered while hot. When the filtrate was gradually cooled to room temperature with stirring, yellow pure berberine chloride was deposited. This is filtered and methanol
After washing with 90 g and drying, 100 g of highly pure berberine chloride (recovery rate: 75% / raw callus-containing berberine standard, purity: 99.5%) was obtained.

Example 2 The same procedure as in Example 1 was carried out except that the slurry of raw callus-0.3N sulfuric acid was not stirred when extracting berberine from the raw callus, but was simply immersed for 4 hours at 25 ° C. in Example 1. The same result as in Example 1 was obtained.

Example 3 The procedure of Example 1 was repeated except that 950 g of concentrated hydrochloric acid was added instead of NaCl in the salt exchange reaction of the extract, and 101 g of purified berberine chloride was obtained (recovery rate: 76%, purity: 99.5%). %) Obtained.

Example 4 The procedure of Example 1 was repeated, except that 1200 g of ethanol was used instead of methanol used for recrystallization of crude berberine chloride, and the obtained purified crystals were washed with 120 g of ethanol. Purified berberine 99g
(Recovery rate 94%, Purity 99.0%) was obtained.

Example 5 The procedure of Example 1 was repeated except that 940 g of 13.4N nitric acid was used in place of adding NaCl to the berberine-containing filtrate, and high-purity berberine nitrate recovery rate was 69.
Obtained in%.

Claims (1)

[Claims] 1. A formula (I) Berberine represented by (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄
= -OCH ₃ ), palmatin (R ₁ , R ₂ , R ₃ , R ₄ = -OC
H ₃ ), Copteisine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ , R ₄ =-
OCH ₂ O-), Jatrolidine (R ₁ = -OH, R ₂ , R ₃ , R ₄
= -OCH ₃ ), Columbamine (R ₁ , R ₃ , R ₄ = -OCH ₃ , R ₂
= -OH), salif engine (R ₁ , R ₂ = -OCH ₂ O-, R ₃ =-
OCH ₃ , R ₄ = -OH) is added to the extract obtained by treating plant tissue pieces or cells containing an isoquinoline alkaloid selected from OCH ₃ and R ₄ = -OH with sulfuric acid, and a salt exchange agent containing chloride ion and / or nitrate ion. A method for separating and purifying an isoquinoline alkaloid, which comprises depositing an isoquinoline alkaloid as a salt of the ion and separating the salt.